Separation of fluorescence-labelled terminal restriction fragment DNA on a two-dimensional gel (T-RFs-2D) - an efficient approach for microbial consortium characterization

Fingerprinting techniques provide access to understanding the ecology of uncultured microbial consortia. However, the application of current techniques such as terminal restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE) has been hindered due to their limitations in characterizing complex microbial communities. This is due to that different populations possibly share the same terminal restriction fragments (T-RFs) and DNA fragments may co-migrate on DGGE gels. To overcome these limitations, a new approach was developed to separate terminal restriction fragments (T-RFs) of 16S rRNA genes on a two-dimensional gel (T-RFs-2D). T-RFs-2D involves restriction digestion of terminal fluorescence-labelled PCR amplified 16S rRNA gene products and their high-resolution separation via a two-dimensional (2D) gel electrophoresis based on the T-RF fragment size (1(st) D) and its sequence composition on the denaturing gradient gel (2(nd) D). The sequence information of interested T-RFs on 2D gels can be obtained through serial poly(A) tailing reaction, PCR amplification and subsequent DNA sequencing. By employing the T-RFs-2D method, bacteria with MspI digested T-RF size of 436 (±1) bp and 514 (±1) bp were identified to be a Lysobacter sp. and a Dehalococcoides sp. in a polychlorinated biphenyl (PCB) dechlorinating culture. With the high resolution of 2D separation, T-RFs-2D separated 63 DNA fragments in a complex river-sediment microbial community, while traditional DGGE detected only 41 DNA fragments in the same sample. In all, T-RFs-2D has its advantage in obtaining sequence information of interested T-RFs and also in characterization of complex microbial communities.     

A novel strategy to extract specific phylogenetic sequence information from community T-RFLP

Cultivation-independent analyses of soil microbial community structures are frequently used to describe microbiological soil characteristics. Semi-automated terminal restriction fragment length polymorphism (T-RFLP) analyses yield high-resolution genetic profiles of highly diverse soil microbial communities and hold great potential for use in routine soil quality monitoring. A serious limitation of T-RFLP analyses has been the inability to reliably affiliate observed terminal restriction fragments (T-RF) to phylogenetic groups. In the study presented here, we were able to overcome this limitation of T-RFLP. With a combination of adapter ligation, fragment size selection, and re-amplification with adapter site specific PCR, we were able to isolate a T-RF-fraction of a narrow size-range containing a T-RF that was significantly more abundant in heavy metal amended soils. Cloning the size-selected T-RF fraction allowed for the efficient isolation of clones containing this specific T-RF. Sequence determination and phylogenetic inference in RDP-II affiliated the sequence to unclassified cyanobacteria. Specific primer design and PCR amplification from bulk soil DNA allowed for independent confirmation of the results from bacterial T-RFLP and T-RF cloning. Our results show that specific T-RFs can be efficiently isolated and identified, and that the adapter ligation approach holds great potential for genetic profiling and for identification of community components of interest.     

Formation of pseudo-terminal restriction fragments,a PCR-related bias affecting terminal restriction fragment length polymorphism analysis of microbial community structure

Terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified genes is a widely used fingerprinting technique in molecular microbial ecology. In this study, we show that besides expected terminal restriction fragments (T-RFs), additional secondary T-RFs occur in T-RFLP analysis of amplicons from cloned 16S rRNA genes at high frequency. A total of 50% of 109 bacterial and 78% of 68 archaeal clones from the guts of cetoniid beetle larvae, using MspI and AluI as restriction enzymes, respectively, were affected by the presence of these additional T-RFs. These peaks were called "pseudo-T-RFs" since they can be detected as terminal fluorescently labeled fragments in T-RFLP analysis but do not represent the primary terminal restriction site as indicated by sequence data analysis. Pseudo-T-RFs were also identified in T-RFLP profiles of pure culture and environmental DNA extracts. Digestion of amplicons with the single-strand-specific mung bean nuclease prior to T-RFLP analysis completely eliminated pseudo-T-RFs. This clearly indicates that single-stranded amplicons are the reason for the formation of pseudo-T-RFs, most probably because single-stranded restriction sites cannot be cleaved by restriction enzymes. The strong dependence of pseudo-T-RF formation on the number of cycles used in PCR indicates that (partly) single-stranded amplicons can be formed during amplification of 16S rRNA genes. In a model, we explain how transiently formed secondary structures of single-stranded amplicons may render single-stranded amplicons accessible to restriction enzymes. The occurrence of pseudo-T-RFs has consequences for the interpretation of T-RFLP profiles from environmental samples, since pseudo-T-RFs may lead to an overestimation of microbial diversity. Therefore, it is advisable to establish 16S rRNA gene sequence clone libraries in parallel with T-RFLP analysis from the same sample and to check clones for their in vitro digestion T-RF pattern to facilitate the detection of pseudo-T-RFs.     

Formation of Pseudo-Terminal Restriction Fragments, a PCR-Related Bias Affecting Terminal Restriction Fragment Length Polymorphism Analysis of Microbial Community Structure

Terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified genes is a widely used fingerprinting technique in molecular microbial ecology. In this study, we show that besides expected terminal restriction fragments (T-RFs), additional secondary T-RFs occur in T-RFLP analysis of amplicons from cloned 16S rRNA genes at high frequency. A total of 50% of 109 bacterial and 78% of 68 archaeal clones from the guts of cetoniid beetle larvae, using MspI and AluI as restriction enzymes, respectively, were affected by the presence of these additional T-RFs. These peaks were called “pseudo-T-RFs” since they can be detected as terminal fluorescently labeled fragments in T-RFLP analysis but do not represent the primary terminal restriction site as indicated by sequence data analysis. Pseudo-T-RFs were also identified in T-RFLP profiles of pure culture and environmental DNA extracts. Digestion of amplicons with the single-strand-specific mung bean nuclease prior to T-RFLP analysis completely eliminated pseudo-T-RFs. This clearly indicates that single-stranded amplicons are the reason for the formation of pseudo-T-RFs, most probably because single-stranded restriction sites cannot be cleaved by restriction enzymes. The strong dependence of pseudo-T-RF formation on the number of cycles used in PCR indicates that (partly) single-stranded amplicons can be formed during amplification of 16S rRNA genes. In a model, we explain how transiently formed secondary structures of single-stranded amplicons may render single-stranded amplicons accessible to restriction enzymes. The occurrence of pseudo-T-RFs has consequences for the interpretation of T-RFLP profiles from environmental samples, since pseudo-T-RFs may lead to an overestimation of microbial diversity. Therefore, it is advisable to establish 16S rRNA gene sequence clone libraries in parallel with T-RFLP analysis from the same sample and to check clones for their in vitro digestion T-RF pattern to facilitate the detection of pseudo-T-RFs.     

Evaluation of three different forward primers by terminal restriction fragment length polymorphism analysis for determination of fecal bifidobacterium spp. in healthy subjects

The 27F forward primer is frequently used in 16S rRNA gene libraries and T-RFLP analysis. However, Bifidobacterium spp. were barely detected with this primer in human fecal samples. In this study, fecal microbiota were analyzed using the T-RFLP method with three different forward primers (27F, 35F, and 529F) in conjunction with one reverse primer (1492R). T-RFLP analysis of fecal microbiota using 35F and 529F detected higher proportions of the terminal restriction fragment (T-RF) corresponding to Bifidobacterium spp. than that using 27F. 27F is in imperfect agreement while 35F and 529F are in good concordance with the 16S rRNA gene sequences of Bifidobacterium spp., and the latter primers allowed for the detection of T-RFs of Bifidobacterium spp. in fecal samples from five healthy subjects. The T-RFs presumed to be Bifidobacterium spp. were cloned and sequenced, and found to match the 16S rRNA gene sequences of Bifidobacterium spp. Among the five fecal samples, two samples with low frequencies of T-RFs of Bifidobacterium spp. were detected using these forward primers. This probably reflects a low prevalence of Bifidobacterium spp. in these two samples. Our study emphasizes the importance of selecting a suitable forward primer for detection of Bifidobacterium spp.     

Coupling of Denaturing High-Performance Liquid Chromatography and Terminal Restriction Fragment Length Polymorphism with Precise Fragment Sizing for Microbial Community Profiling and Characterization

Terminal restriction fragment length polymorphism (T-RFLP) is used to monitor the structural diversity of complex microbial communities in terms of richness, relative abundance, and distribution of the major subpopulations and individual members. However, discrepancies of several nucleotides between expected and experimentally observed lengths of terminal restriction fragments (T-RFs), together with the difficulty of obtaining DNA sequence information from T-RFLP profiling, often prevent accurate phylogenetic characterization of the microbial community of interest. In this study, T-RFLP analysis of DNA from an artificial assembly of five bacterial strains was carried out with a combination of two size markers with different fluorescent tags. Precise sizing of T-RFs in the 50- to 500-nucleotide range was achieved by using the same dye for both samples and size markers. Phylogenetic assignment of the component microbial strains was facilitated by coupling T-RFLP to denaturing high-performance liquid chromatography (D-HPLC) of 16S RNA gene fragments followed by direct sequencing. The proposed coupling of D-HPLC and T-RFLP provides unambiguous characterization of microbial communities containing less than 15 microbial strains.Over the last 2 decades, the development of molecular biology tools has led to the emergence of a new discipline, molecular microbial ecology. The overall structural diversity of microbial communities can be examined easily using PCR-based strategies (6), usually targeting the 16S rRNA gene as a universal genetic marker of prokaryotes. Genotyping approaches avoid current limitations of cultivation methods, which only poorly reflect the phylogenetic diversity of microbial communities (12). The principles, technical aspects, and limitations of commonly employed methods were recently reviewed (10). Among these methods, terminal restriction fragment length polymorphism (T-RFLP) has proved to be invaluable for rapid characterization of the composition and dynamics of species-rich samples (13). Compared to other approaches, T-RFLP is semiquantitative and combines high levels of sensitivity, resolution, and reproducibility (see Table S1 in the supplemental material). Taxonomic diversity of microbial communities is evaluated by using the strain-dependent variability of restriction sites within a conserved PCR-amplified DNA fragment. The terminal restriction fragments (T-RFs) of digested PCR products appear as chromatographic peaks after size-dependent electrophoretic separation due to a fluorescent tag attached to one of the primers used for PCR. The relative abundance of peaks is evaluated, and fragment lengths are estimated using a fluorescent internal size standard comigrating with the sample (5). The estimated lengths corresponding to the T-RFLP peaks obtained are compared to databases of T-RF sizes generated by in silico digestion of known 16S rRNA gene sequences with commonly used restriction enzymes for phylogenetic assignment (13). However, estimation of T-RF lengths from experimental chromatograms is biased by the fact that differences in the electrophoretic properties of the two different fluorescent dyes used to distinguish sample fragments from the size marker significantly affect fragment migration (7, 11). Discrepancies greater than 6 nucleotides (nt), depending on the length of the fragment, have been reported between expected and experimentally estimated fragment lengths (7). This causes errors in phylogenetic assignments and may in turn lead to erroneous inferences regarding the functional aspects of the microbial communities under investigation. Another drawback of T-RFLP is the difficulty of retrieving sequence information directly from experimental T-RFs, since additional construction of representative 16S rRNA gene libraries is required to obtain such information.Here we propose an experimental strategy to circumvent current limitations of T-RFLP and facilitate characterization of microbial communities. First, we propose an optimized protocol for T-RFLP that yields reliable T-RF sizes. Second, we describe use of denaturing high-performance liquid chromatography (D-HPLC) as an alternative to cloning in order to gain direct access to DNA sequence information. D-HPLC, an emerging technique for microbial community profiling (1, 4), enables collection of DNA fragments separated on the basis of differences in sequence, sequence length, and G+C content at a partially denaturing temperature. The unambiguous phylogenetic characterization of a model microbial assembly of five reference strains is described as proof of principle of the usefulness of the proposed strategy.     

Impact of T-RFLP data analysis choices on assessments of microbial community structure and dynamics

Background

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a common DNA-fingerprinting technique used for comparisons of complex microbial communities. Although the technique is well established there is no consensus on how to treat T-RFLP data to achieve the highest possible accuracy and reproducibility. This study focused on two critical steps in the T-RFLP data treatment: the alignment of the terminal restriction fragments (T-RFs), which enables comparisons of samples, and the normalization of T-RF profiles, which adjusts for differences in signal strength, total fluorescence, between samples.

Results

Variations in the estimation of T-RF sizes were observed and these variations were found to affect the alignment of the T-RFs. A novel method was developed which improved the alignment by adjusting for systematic shifts in the T-RF size estimations between the T-RF profiles. Differences in total fluorescence were shown to be caused by differences in sample concentration and by the gel loading. Five normalization methods were evaluated and the total fluorescence normalization procedure based on peak height data was found to increase the similarity between replicate profiles the most. A high peak detection threshold, alignment correction, normalization and the use of consensus profiles instead of single profiles increased the similarity of replicate T-RF profiles, i.e. lead to an increased reproducibility. The impact of different treatment methods on the outcome of subsequent analyses of T-RFLP data was evaluated using a dataset from a longitudinal study of the bacterial community in an activated sludge wastewater treatment plant. Whether the alignment was corrected or not and if and how the T-RF profiles were normalized had a substantial impact on ordination analyses, assessments of bacterial dynamics and analyses of correlations with environmental parameters.

Conclusions

A novel method for the evaluation and correction of the alignment of T-RF profiles was shown to reduce the uncertainty and ambiguity in alignments of T-RF profiles. Large differences in the outcome of assessments of bacterial community structure and dynamics were observed between different alignment and normalization methods. The results of this study can therefore be of value when considering what methods to use in the analysis of T-RFLP data.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-014-0360-8) contains supplementary material, which is available to authorized users.     

Application of terminal restriction fragment length polymorphism (T-RFLP) analysis to monitor effect of biocontrol agents on rhizosphere microbial community of hot pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.)

Microbial communities in hot pepper (Capsicum annuum L.) cultivation fields under different cultivation methods were investigated by terminal restriction fragment length polymorphism (T-RFLP) analysis. Rhizosphere soil and leaf samples were collected from control, conventional and nature-friendly cultivation fields between May and July, 2009. Two Bacillus subtilis strains were applied to nature-friendly cultivation fields as biocontrol agents during the sampling period. Relative abundances of bacteria and plant pathogenic fungi related T-RFs were also measured to monitor the effect of biocontrol agents on potential plant pathogenic fungi. In the principal component analysis (PCA) based on T-RFLP profiles, the microbial communities from rhizosphere soil samples in July, including bacteria and fungi, showed distinct difference between nature-friendly cultivation fields and other cultivation fields. However, there was no correlation between cultivation methods and leaf microbial communities at any sampling period. Changes in the abundance of bacteria related T-RF in the rhizosphere of nature-friendly cultivation fields were observed clearly two months after application of biocontrol agent, while the abundance of plant pathogenic fungi related T-RFs significantly decreased.     

Reliability for detecting composition and changes of microbial communities by T-RFLP genetic profiling

Terminal restriction fragment length polymorphism (T-RFLP) analysis is commonly used for profiling microbial communities in various environments. However, it may suffer from biases during the analytic process. This study addressed the potential of T-RFLP profiles (1) to reflect real community structures and diversities, as well as (2) to reliably detect changing components of microbial community structures. For this purpose, defined artificial communities of 30 SSU rRNA gene clones, derived from nine bacterial phyla, were used. PCR amplification efficiency was one primary bias with a maximum variability factor of 3.5 among clones. PCR downstream analyses such as enzymatic restriction and capillary electrophoresis introduced a maximum bias factor of 4 to terminal restriction fragment (T-RF) signal intensities, resulting in a total maximum bias factor of 14 in the final T-RFLP profiles. In addition, the quotient between amplification efficiency and T-RF size allowed predicting T-RF abundances in the profiles with high accuracy. Although these biases impaired detection of real community structures, the relative changes in structures and diversities were reliably reflected in the T-RFLP profiles. These data support the suitability of T-RFLP profiling for monitoring effects on microbial communities.     

Web-Based Phylogenetic Assignment Tool for Analysis of Terminal Restriction Fragment Length Polymorphism Profiles of Microbial Communities

Culture-independent DNA fingerprints are commonly used to assess the diversity of a microbial community. However, relating species composition to community profiles produced by community fingerprint methods is not straightforward. Terminal restriction fragment length polymorphism (T-RFLP) is a community fingerprint method in which phylogenetic assignments may be inferred from the terminal restriction fragment (T-RF) sizes through the use of web-based resources that predict T-RF sizes for known bacteria. The process quickly becomes computationally intensive due to the need to analyze profiles produced by multiple restriction digests and the complexity of profiles generated by natural microbial communities. A web-based tool is described here that rapidly generates phylogenetic assignments from submitted community T-RFLP profiles based on a database of fragments produced by known 16S rRNA gene sequences. Users have the option of submitting a customized database generated from unpublished sequences or from a gene other than the 16S rRNA gene. This phylogenetic assignment tool allows users to employ T-RFLP to simultaneously analyze microbial community diversity and species composition. An analysis of the variability of bacterial species composition throughout the water column in a humic lake was carried out to demonstrate the functionality of the phylogenetic assignment tool. This method was validated by comparing the results generated by this program with results from a 16S rRNA gene clone library.     

Web-based phylogenetic assignment tool for analysis of terminal restriction fragment length polymorphism profiles of microbial communities

Culture-independent DNA fingerprints are commonly used to assess the diversity of a microbial community. However, relating species composition to community profiles produced by community fingerprint methods is not straightforward. Terminal restriction fragment length polymorphism (T-RFLP) is a community fingerprint method in which phylogenetic assignments may be inferred from the terminal restriction fragment (T-RF) sizes through the use of web-based resources that predict T-RF sizes for known bacteria. The process quickly becomes computationally intensive due to the need to analyze profiles produced by multiple restriction digests and the complexity of profiles generated by natural microbial communities. A web-based tool is described here that rapidly generates phylogenetic assignments from submitted community T-RFLP profiles based on a database of fragments produced by known 16S rRNA gene sequences. Users have the option of submitting a customized database generated from unpublished sequences or from a gene other than the 16S rRNA gene. This phylogenetic assignment tool allows users to employ T-RFLP to simultaneously analyze microbial community diversity and species composition. An analysis of the variability of bacterial species composition throughout the water column in a humic lake was carried out to demonstrate the functionality of the phylogenetic assignment tool. This method was validated by comparing the results generated by this program with results from a 16S rRNA gene clone library.     

TRiFLe, a Program for In Silico Terminal Restriction Fragment Length Polymorphism Analysis with User-Defined Sequence Sets

We describe TRiFLe, a freely accessible computer program that generates theoretical terminal restriction fragments (T-RFs) from any user-supplied sequence set tailored to a particular group of organisms, sequences from clone libraries, or sequences from specific genes. The program allows a rapid identification of the most polymorphic enzymes, creates a collection of T-RFs for the data set, and can potentially identify specific T-RFs in T-RF length polymorphism (T-RFLP) patterns by comparing theoretical and experimental results. TRiFLE was used for analyzing T-RFLP data generated for the amoA and pmoA genes. The peaks identified in the T-RFLP patterns show an overlap of ammonia- and methane-oxidizing bacteria in the metalimnion of a subtropical lake.     

Novel phylogenetic assignment database for terminal-restriction fragment length polymorphism analysis of human colonic microbiota

Various molecular-biological approaches using the 16S rRNA gene sequence have been used for the analysis of human colonic microbiota. Terminal- restriction fragment length polymorphism (T-RFLP) analysis is suitable for a rapid comparison of complex bacterial communities. Terminal-restriction fragment (T-RF) length can be calculated from a known sequence, thus one can predict bacterial species on the basis of their T-RF length by this analysis. The aim of this study was to build a phylogenetic assignment database for T-RFLP analysis of human colonic microbiota (PAD-HCM), and to demonstrate the effectiveness of PAD-HCM compared with the results of 16S rRNA gene clone library analysis. PAD-HCM was completed to include 342 sequence data obtained using four restriction enzymes. Approximately 80% of the total clones detected by 16S rRNA gene clone library analysis were the same bacterial species or phylotypes as those assigned from T-RF using PAD-HCM. Moreover, large T-RFs consisted of common species or phylotypes detected by both analytical methods. All pseudo-T-RFs identified by mung bean nuclease digestion could not be assigned to a bacterial species or phylotype, and this finding shows that pseudo-T-RFs can also be predicted using PAD-HCM. We conclude that PAD-HCM built in this study enables the prediction of T-RFs at the species level including difficult-to-culture bacteria, and that it is very useful for the T-RFLP analysis of human colonic microbiota.     

Evaluation of terminal-restriction fragment length polymorphism analysis in contrasting marine environments

Terminal-restriction fragment length polymorphism (T-RFLP) analysis is widely used in microbial ecology studies. In the present study, T-RFLP analysis of PCR products digested by five restriction enzymes (AluI, HaeIII, MspI, Sau3AI and TaqI) was applied for 20 samples from three contrasting coastal environments to assess the biases associated with the choice of enzyme digestion and T-RF analysis. The five enzyme digestions produced highly variable species richness (in terms of number of T-RFs). Analysis of peak areas with a threshold of 0.5% of the total peak area, which recovered 92-96% of the total peak area, revealed different diversity indexes from the five enzyme digestions. Multidimensional scaling, based on matrices that were generated by scoring peak presence/absence and area, revealed similar bacterial community structure patterns among the 20 samples, regardless of the choice of restriction enzymes. Our results strongly argue that the choice of different digestion enzymes in the T-RFLP technique generated valid and consistent bacterial community structures but highly variable species richness and diversity indices. The biases associated with the choice of digestion enzymes needs to be evaluated carefully or at least to be addressed when using T-RFLP analysis.     

Increase in terminal restriction fragments of Bacteroidetes-derived 16S rRNA genes after administration of short-chain fructooligosaccharides

It is well known that short chain fructooligosaccharides (scFOS) modify intestinal microbiota in animals as well as in humans. Since most murine intestinal bacteria are still uncultured, it is difficult for a culturing method to detect changes in intestinal microbiota after scFOS administration in a mouse model. In this study, we sought markers of positive change in murine intestinal microbiota after scFOS administration using terminal restriction fragment length polymorphism (T-RFLP) analysis, which is a culture-independent method. The T-RFLP profiles showed that six terminal restriction fragments (T-RFs) were significantly increased after scFOS administration. Phylogenetic analysis of the 16S rRNA partial gene sequences of murine fecal bacteria suggested that four of six T-RFs that increased after scFOS administration were derived from the 16S rRNA genes of the class Bacteroidetes. Preliminary quantification of Bacteroidetes by real-time PCR suggests that the 16S rRNA genes derived from Bacteroidetes were increased by scFOS administration. Therefore, the T-RFs derived from Bacteroidetes are good markers of change of murine intestinal microbiota after scFOS administration.     

Development of a fluorophore-ribosomal DNA restriction typing method for monitoring structural shifts of microbial communities

DNA restriction fragment polymorphism technologies such as amplified ribosomal DNA restriction analysis (ARDRA) and terminal restriction fragment length polymorphism (T-RFLP) have been widely used in investigating microbial community structures. However, these methods are limited due to either the low resolution or sensitivity. In this study, a fluorophore-ribosomal DNA restriction typing (f-DRT) approach is developed for structural profiling of microbial communities. 16S rRNA genes are amplified from the community DNA and digested by a single restriction enzyme Msp I. All restriction fragments are end-labeled with a fluorescent nucleotide Cy5-dCTP via a one-step extension reaction and detected with an automated DNA sequencer. All 50 predicted restriction fragments between 100 and 600 bp were detected when twelve single 16S rRNA gene sequences were analyzed using f-DRT approach; 92% of these fragments were determined with accuracy of ±2 bp. In the defined model communities containing five components with different ratios, relative abundance of each component was correctly revealed by this method. The f-DRT analysis also showed structural shifts of intestinal microbiota in carcinogen-treated rats during the formation of precancerous lesions in the colon, as sensitive as multiple digestion-based T-RFLP analysis. This study provides a labor and cost-saving new method for monitoring structural shifts of microbial communities.     

Use of the T-RFLP technique to assess spatial and temporal changes in the bacterial community structure within an agricultural soil planted with transgenic and non-transgenic potato plants

The aim of this study was to examine whether the terminal restriction fragment length polymorphism (T-RFLP) analysis represents an appropriate technique for monitoring highly diverse soil bacterial communities, i.e. to assess spatial and/or temporal effects on bacterial community structure. The T-RFLP method, a recently described fingerprinting technique, is based on terminal restriction fragment length polymorphisms between distinct small-subunit rRNA gene sequence types. This technique permits an automated quantification of the fluorescence signal intensities of the individual terminal restriction fragments (T-RFs) in a given community fingerprint pattern. The indigenous bacterial communities of three soil plots located within an agricultural field of 110 m(2) were compared. The first site was planted with non-transgenic potato plants, while the other two were planted with transgenic GUS and Barnase/Barstar potato plants, respectively. Once prior to planting and three times after planting, seven parallel samples were taken from each of the three soil plots. The T-RFLP analysis resulted in very complex but highly reproducible community fingerprint patterns. The percentage abundance values of defined T-RFs were calculated for the seven parallel samples of the respective soil plot. A multivariate analysis of variance was used to test T-RFLP data sets for significant differences. The statistical treatments clearly revealed spatial and temporal effects, as well as spacextime interaction effects, on the structural composition of the bacterial communities. T-RFs which showed the highest correlations to the discriminant factors were not those T-RFs which showed the largest single variations between the seven-sample means of individual plots. In summary, the T-RFLP technique, although a polymerase chain reaction-based method, proved to be a suitable technique for monitoring highly diverse soil microbial communities for changes over space and/or time.     

Movement and Fixation of Intestinal Microbiota after Administration of Human Feces to Germfree Mice

Human flora-associated (HFA) mice have been considered a tool for studying the ecology and metabolism of intestinal bacteria in humans, although they have some limitations as a model. Shifts in dominant species of microbiota in HFA mice after the administration of human intestinal microbiota was revealed by 16S rRNA gene sequence and terminal restriction fragment length polymorphism (T-RFLP) analyses. Characteristic terminal restriction fragments (T-RFs) were quantified as the proportion of total peak area of all T-RFs. Only the proportion of the T-RF peak at bp 366, identified as the Gammmaproteobacteria group and the family Coriobacteriaceae, was reduced in this study. Increased T-RFs over time at bp 56, 184, and 196 were affiliated with the Clostridium group. However, most of the isolated bacteria with unique population shifts were phylotypes. The vertical transmission of the intestinal microbiota of the mouse offspring was also investigated by dendrogram analysis derived from the similarity of T-RFLP patterns among samples. As a result, the intestinal microbiota of HFA mice and their offspring reflected the composition of individual human intestinal bacteria with some modifications. Moreover, we revealed that human-derived lactobacilli (HDL), which have been considered difficult to colonize in the HFA mouse intestine in previous studies based on culture methods, could be detected in the HFA mouse intestine by using a lactic acid bacterium-specific primer and HDL-specific primers. Our results indicate that the intestinal microbiota of HFA mice represents a limited sample of bacteria from the human source and are selected by unknown interactions between the host and bacteria.     

Application of a new PCR primer for terminal restriction fragment length polymorphism analysis of the bacterial communities in plant roots

Contamination with plastid small subunit (SSU) rDNA is a major drawback when analyzing the bacterial communities of plant roots using culture-independent methods. In this study, a polymerase chain reaction (PCR) primer, 783r, was designed and tested to specifically amplify the SSU rDNA of various bacterial species without amplifying the SSU rDNA of plant plastids. To confirm how useful the community analysis of rhizobacteria is using 783r, the terminal restriction fragment length polymorphism (T-RFLP) method was performed with wheat (Triticum aestivum) and spinach (Spinacea oleracea) root samples. Using the standard T-RFLP method, a large T-RF peak of plant plastid SSU rDNA interfered with the bacterial community analysis. In contrast, the T-RFLP method using the 783r primer was able to detect the bacterial DNA while directly eliminating the influence of the plant-derived DNA extracted from the plant roots. Primer 783r might, therefore, be a useful PCR primer for the culture-independent analysis of bacterial communities in plant roots using SSU rDNA.